Interpretation of cytochrome P450 monooxygenase kinetics by modeling of thermodynamic activity.

The experimentally determined Michaelis constant K results from a combination of two effects: the recognition of the substrate by the enzyme and the molecular interactions between substrate and solvent. By separating substrate recognition from solvent effects, the thermodynamic activity-based Michaelis constant K allows for an unambiguous comparison of how different substrates fit into the substrate binding site. K of a poorly water-soluble substrate is calculated from the experimentally determined concentration-based Michaelis constant K and its activity coefficient at infinite dilution γ. Comparing the K of different substrates instead of the experimentally determined K prevents misinterpretations of the molecular basis of enzyme-substrate interactions. While n-octane showed the lowest K value of six P450 substrates, its K was 500 fold higher than aniline, indicating that the binding of n-octane is mainly driven by its low water solubility, while binding of aniline is driven by its shape complementarity. For three substrates (aniline, oct-1-yne, n-octane), γ was reliably calculated by molecular dynamics simulations, either in binary substrate-water mixtures or in ternary mixtures including DMSO as cosolvent. It is demonstrated that the widely used DMSO has a considerable effect on the measured K value. Depending on the substrate, addition of 10% v/v DMSO increases K by up to a factor of 11. To make biocatalytic experiments reproducible, it is therefore of utmost importance to carefully report the reaction conditions. The reliable simulation of activity coefficients in complex mixtures allows for an unambiguous comparison of enzyme-substrate interactions and provides a predictive tool for the design of biocatalytic processes.